Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tumor necrosis factor receptor 1 |
| Abbreviated Name | TNF-R1 |
| SCOP Family | TNF receptor-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P19438 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | extracellular domain only expressed and refolded (aa.40-201) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 162 |
| Molecular Weight | 18311.7 |
| Pi | 6.95904 |
| Molecular Weight | 18311.7 |
| Disulphides | >10 |
| Full Sequence |
MDSVCPQGKYI HPQNNSICCT KCHKGTYLYN DCPGPGQDTD CRECESGSFT ASENHLRHCL SCSKCRKEMG QVEISSCTVD RDTVCGCRKN QYRHYWSENL FQCFNCSLCL NGTVHLSCQE KQNTVCTCHA GFFLRENECV SCSNCKKSLE CTKLCLPQIEN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hale KK, Smith CG, Baker SL, Vanderslice RW, Squires CH, Gleason TM, Tucker KK, Kohno T, Russell DA (1995) Cytokine, 7, 26-38 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pT3X1-2 |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | not stated |
| Solubilization Buffer | 6M GdnHCl, 100mM TrisHCl, 4mM PMSF, pH 8.5 |
| Refolding Buffer | 50mM TrisHCl pH 10.7 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 10.7 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 16h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 6mM |
| Refolding Protocol | Protein was isolated and washed inclusion bodies was solubilized in Solubilization buffer for 1h at room temperature. The soultion was then reduced for 30min with 20mM DTT. The mixture was then diluted 10-fold with refolding buffer, 6mM cysteine and 4mM PMSF were also added. The protein was allowed to refold at 4degC for 16h, before centrifugation (20min, 20000g) to remove insoluble material. The supernatant was dialysed against 50mM TrisHCl pH 7.5 and centrifuged again. 4mM PMSF was added to the supernatant, which was then loaded onto a TNF-alpha-affinity column. After elution, the protein was further purified by reverse phase FPLC |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | similar protocol for TNF-R2 |